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A B S T R A C T PurposeWe investigated the global gene expression in a large panel of pancreatic endocrine tumors (PETs) aimed at identifying new potential targets for therapy and biomarkers to predict patient outcome. Patients and MethodsUsing a custom microarray, we analyzed 72 primary PETs, seven matched metastases, and 10 normal pancreatic samples. Relevant differentially expressed genes were validated by either quantitative real-time polymerase chain reaction or immunohistochemistry on tissue microarrays. ResultsOur data showed that: tuberous sclerosis 2 (TSC2) and phosphatase and tensin homolog (PTEN) were downregulated in most of the primary tumors, and their low expression was significantly associated with shorter disease-free and overall survival; somatostatin receptor 2 (SSTR2) was absent or very low in insulinomas compared with nonfunctioning tumors; and expression of fibroblast growth factor 13 (FGF13) gene was significantly associated with the occurrence of liver metastasis and shorter disease-free survival. TSC2 and PTEN are two key inhibitors of the Akt/mammalian target of rapamycin (mTOR) pathway and the specific inhibition of mTOR with rapamycin or RAD001 inhibited cell proliferation of PET cell lines. ConclusionOur results strongly support a role for PI3K/Akt/mTOR pathway in PET, which ties in with the fact that mTOR inhibitors have reached phase III trials in neuroendocrine tumors. The finding of differential SSTR expression raises the potential for SSTR expression to be evaluated as a marker of response to somatostatin analogs. Finally, we identified FGF13 as a new prognostic marker that predicted poorer outcome in patients who were clinically considered free from disease.

The three mammalian bombesin (Bn) receptors (gastrin-releasing peptide [GRP] receptor, neuromedin B [NMB] receptor, BRS-3) are one of the classes of G protein-coupled receptors that are most frequently over-express/ectopically expressed by common, important malignancies. Because of the clinical success of somatostatin receptor-mediated imaging and cytotoxicity with neuroendocrine tumors, there is now increasing interest in pursuing a similar approach with Bn receptors. In the last few years then have been more than 200 studies in this area. In the present paper, the in vitro and in vivo results, as well as results of human studies from many of these studies are reviewed and the current state of Bn receptor-mediated imaging or cytotoxicity is discussed. Both Bn receptor-mediated imaging studies as well as Bn receptor-mediated tumoral cytotoxic studies using radioactive and non-radioactive Bn-based ligands are covered. Keywordsbombesin; gastrin-releasing peptide; neuromedin B; BRS-3; receptor-mediated imaging; tumor cytotoxicity; DOTA; DTPA; NOTA I. Bombesin (Bn) receptor family-General (Table 1,2)The mammalian Bn receptor family is receiving increased attention as a means of localizing tumors or other disease processes by receptor-mediated imaging or for receptor-mediated cytotoxicity of tumors [1][2][3][4][5]. This family got its unusual name, because the original members of this peptide family were isolated from various frog skins and were named after the frog they were isolated from, with the original amidated tetradecapeptide isolated from the European frog, Bombina bombina in 1970 [6][7][8] (Table 2). Subsequently, a large number of related peptides were isolated which were divided into three groups: the Bn-related peptides with a COOH terminal, Gly-His-Leu-Met-NH 2 , the ranatesin-litorin group with a COOH terminus of Gly-His-Phe-Met-NH 2 and the phyllolitorin group with a COOH terminus ending in Gly-Ser-Phe/Leu-Met-NH 2 (Table 2) [6][7][8]. Subsequently two mammalian equivalent peptides were isolated, gastrin-releasing peptide (GRP), a 27 amino Corresponding author: Dr R.T. Jensen, Building 10, Room 9C-103, National Institutes of Health, Bethesda, MD 20892, Phone-301-496-4201, Fax-301-402-0600, robertj@bdg10.niddk.nih.gov. * Both authors contributed equally to this work Conflict of Interest: None NIH Public Access Author ManuscriptCurr Drug Deliv. Author manuscript; available in PMC 2012 January 1. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript acid peptide which shares the same seven COOH terminal amino acids with Bn (Table 2) [9] and the decapeptide, neuromedin B (NMB) (Tables 1,2) which shares 6 of the 7 COOH terminal amino acids with litorin (Table 2) [10]. Each of these peptides is widely distributed in both the central nervous system (CNS) and peripheral tissues, especially in the gastrointestinal (GI) tract [8]. Numerous studies demonstrate these two peptides are involved in a wide range of physiological and pathophysiological processes which include: in the CNS...

The effects of bombesin receptor subtype-3 (BRS-3) agonists were investigated on lung cancer cells. The BRS-3 agonist (DTyr6, βAla11, Phe13, Nle14)bombesin6-14 (BA1), but not gastrin releasing peptide (GRP) or neuromedin B (NMB) increased significantly the clonal growth of NCI-H1299 cells stably transfected with BRS-3 (NCI-H1299-BRS-3). Also, BA1 addition to NCI-H727 or NCI-H1299-BRS-3 cells caused Tyr1068 phosphorylation of the epidermal growth factor receptor (EGFR). Similarly, (DTyr6, R-Apa11, Phe13, Nle14)bombesin6-14 (BA2) and (DTyr6, R-Apa11, 4-Cl,Phe13, Nle14)bombesin6-14 (BA3) but not gastrin releasing peptide (GRP) or neuromedin B (NMB) caused EGFR transactivation in NCI-H1299-BRS-3 cells. BA1-induced EGFR or ERK tyrosine phosphorylation was not inhibited by addition of BW2258U89 (BB2R antagonist) or PD168368 (BB1R antagonist) but was blocked by (DNal-Cys-Tyr-DTrp-Lys-Val-Cys-Nal)NH2 (BRS-3 ant.). The BRS-3 ant. reduced clonal growth of NCI-H1299-BRS-3 cells. BA1, BA2, BA3 and BRS-3 ant. inhibit specific 125I-BA1 binding to NCI-H1299-BRS-3 cells with an IC50 values of 1.1, 21, 15 and 750 nM respectively. The ability of BRS-3 to regulate EGFR transactivation in NCI-H1299-BRS-3 cells was reduced by AG1478 or gefitinib (EGFR tyrosine kinase inhibitors), GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor), N-acetylcysteine (anti-oxidant), Tiron (superoxide scavenger) and DPI (NADPH oxidase inhibitor). These results demonstrate that BRS-3 agonists may stimulate lung cancer growth as a result of EGFR transactivation and that the transactivation is regulated by BRS-3 in a Src-, reactive oxygen and matrix metalloprotease-dependent manner.

Pancreatic endocrine tumours (PETs) are rare and 'indolent' neoplasms that usually develop metastatic lesions and exhibit poor response to standard medical treatments. Few studies have investigated pathways responsible for PET cell growth and invasion and no alternative therapeutic strategies have been proposed. In a recent microarray analysis for genes up-regulated in PETs, we have described the up-regulation of soluble Src family tyrosine kinases in this neoplasia, which may represent potentially promising candidates for therapy. Herein, we have investigated the expression and function of Src family kinases in PETS and PET cell lines. Western blot analysis indicated that Src is highly abundant in the PET cell lines CM and QGP-1. Immunohistochemistry and Western blot analyses showed that Src is up-regulated also in human PET lesions. Pharmacological inhibition of Src family kinases by the specific inhibitor PP2 strongly interfered with adhesion, spreading and migration of PET cell lines. Accordingly, the actin cytoskeleton was profoundly altered after inhibition of Src kinases, whereas even prolonged incubation with PP2 exerted no effect on cell cycle progression and/or apoptosis of PET cells. A transient increase in tyrosine phosphorylation of a subset of proteins was observed in QGP-1 cells adhering to the plate, with a peak at 75 min after seeding, when approximately 80% of cells were attached. Inhibition of Src kinases caused a dramatic reduction in the phosphorylation of proteins with different molecular weight that were isolated from the cell extracts by anti-phosphotyrosine immunoprecipitation or pull-down with the SH2 domain of Src. Among them, the docking protein p130Cas interacted with Src and is a major substrate of the Src kinases in QGP-1 cells undergoing adhesion. Our results suggest that Src kinases play a specific role during adhesion, spreading and migration of PET cells and may indicate therapeutical approaches directed to limiting the metastatic potential of these cells.